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Muscle stem cells (MuSCs) are essential for the growth, maintenance, and repair of skeletal muscle. In the emerging area of cultured meat, meat products are manufactured with MuSCs using theory and technology from the fields of cell culture, tissue engineering, and food processing. Recently, considerable progress has been made in bioprocessing technologies for MuSCs, including isolation, expansion, differentiation, and tissue building. Here we summarize cutting-edge operational strategies and recently characterized regulatory mechanisms for MuSCs. Furthermore, we discuss their applicability to refining the production process for cultured meat and accelerating its industrialization.

The hydroxylation is an important way to generate the functionalized derivatives of flavonoids. However, the efficient hydroxylation of flavonoids by bacterial P450 enzymes is rarely reported. Here, a bacterial P450 sca-2mut whole-cell biocatalyst with an outstanding 3′-hydroxylation activity for the efficient hydroxylation of a variety of flavonoids was first reported. The whole-cell activity of sca-2mut was enhanced using a novel combination of flavodoxin Fld and flavodoxin reductase Fpr from Escherichia coli. In addition, the double mutant of sca-2mut (R88A/S96A) exhibited an improved hydroxylation performance for flavonoids through the enzymatic engineering. Moreover, the whole-cell activity of sca-2mut (R88A/S96A) was further enhanced by the optimization of whole-cell biocatalytic conditions. Finally, eriodictyol, dihydroquercetin, luteolin, and 7,3′,4′-trihydroxyisoflavone, as examples of flavanone, flavanonol, flavone, and isoflavone, were produced by whole-cell biocatalysis using naringenin, dihydrokaempferol, apigenin, and daidzein as the substrates, with the conversion yield of 77%, 66%, 32%, and 75%, respectively. The strategy used in this study provided an effective method for the further hydroxylation of other high value-added compounds.

Hemoglobin (Hb) and myoglobin (Mb) are kinds of heme-binding proteins that play crucial physiological roles in different organisms. With rapid application development in food processing and biocatalysis, the requirement of biosynthetic Hb and Mb is increasing. However, the production of Hb and Mb is limited by the lower expressional level of globins and insufficient or improper heme supply. After selecting an inducible strategy for the expression of globins, removing the spatial barrier during heme synthesis, increasing the synthesis of 5-aminolevulinate and moderately enhancing heme synthetic rate-limiting steps, the microbial synthesis of bovine and porcine Hb was firstly achieved. Furthermore, an engineered Saccharomyces cerevisiae obtained a higher titer of soybean (108.2 ± 3.5 mg/L) and clover (13.7 ± 0.5 mg/L) Hb and bovine (68.9 ± 1.6 mg/L) and porcine (85.9 ± 5.0 mg/L) Mb. Therefore, this systematic engineering strategy will be useful to produce other hemoproteins or hemoenzymes with high activities.

Metabolic heterogeneity and dynamic changes in metabolic fluxes are two inherent characteristics of microbial fermentation that limit the precise control of metabolisms, often leading to impaired cell growth and low productivity. Dynamic metabolic engineering addresses these challenges through the design of multi-layered and multi-genetic dynamic regulation network (DRN) that allow a single cell to autonomously adjust metabolic flux in response to its growth and metabolite accumulation conditions. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2S)-naringenin and p-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In this scenario, the acyl carrier protein was used as a novel critical node for fine-tuning malonyl-CoA consumption instead of direct repression of fatty acid synthase commonly employed in previous studies. To do so, we first engineered a multi-layered DRN enabling single cells to concurrently regulate acpH, acpS, acpT, acs, and ACC in malonyl-CoA catabolic and anabolic pathways. Next, the NCOMB DRN was optimized to enhance the synergies between different dynamic regulation layers via a biosensor-based directed evolution strategy. Finally, a high producer obtained from NCOMB DRN approach yielded a 8.7-fold improvement in (2S)-naringenin production (523.7 ± 51.8 mg/L) with a concomitant 20% increase in cell growth compared to the base strain using static strain engineering approach, thus demonstrating the high efficiency of this system for improving pathway production.

Gluconobacter oxydans is an important industrial bacterial strain widely used to produce a lot of useful products. However, very few gene editing tools are available for G. oxydans. This study aimed to develop an efficient genome editing method for G. oxydans using SacB as a counter-selectable marker. A plasmid that could express the kanamycin resistance gene in both E. coli and G. oxydans was constructed using the screened shuttle promoter P116. After optimizing the genome editing conditions, the derivative plasmids could be effectively utilized for diverse genome editing, such as gene deletion, insertion, replacement, and in situ modification in G. oxydans WSH-003. In addition, the SacB-based system also achieved multiple gene editing in G. oxydans. Moreover, the genome of the industrial strain G. oxydans WSH-003 was modified and the growth rate and substrate conversion rate of the strain successfully increased using this system. The system could also have potential to be applied in different G. oxydans strains. The process established in this study also provides a reference for constructing genetic tools for many other genetically recalcitrant bacteria.

The corpus luteum (CL) is a temporary organ that plays a critical role for female fertility by maintaining the estrous cycle. MicroRNA (miRNA) is a class of non-coding RNAs involved in various biological processes. However, there exists limited knowledge of the role of miRNA in yak CL. In this study, we used high-throughput sequencing to study the transcriptome dynamics of miRNA in yak early (eCL), middle (mCL) and late-stage CL (lCL). A total of 6,730 miRNAs were identified, including 5,766 known and 964 novels miRNAs. Three miRNAs, including bta-miR-126-3p, bta-miR-143 and bta-miR-148a, exhibited the highest expressions in yak CLs of all the three stages. Most of the miRNAs were 20-24 nt in length and the peak was at 22 nt. Besides, most miRNAs with different lengths displayed significant uracil preference at the 5'-end. Furthermore, 1,067, 280 and 112 differentially expressed (DE) miRNAs were found in eCL vs. mCL, mCL vs. lCL, and eCL vs. lCL, respectively. Most of the DE miRNAs were down-regulated in the eCL vs. mCL and eCL vs. lCL groups, and up-regulated in the mCL vs. lCL group. A total of 18,904 target genes were identified, with 18,843 annotated. Pathway enrichment analysis of the DE miRNAs target genes illustrated that the most enriched cellular process in each group included pathways in cancer, PI3K-Akt pathway, endocytosis, and focal adhesion. A total of 20 putative target genes in 47 DE miRNAs were identified to be closely associated with the formation, function or regression of CL. Three DE miRNAs, including bta-miR-11972, novel-miR-619 and novel-miR-153, were proved to directly bind to the 3'-UTR of their predicated target mRNAs, including CDK4, HSD17B1 and MAP1LC3C, respectively. Both of these DE miRNAs and their target mRNAs exhibited dynamic expression profiles across the lifespan of yak CL. This study presents a general basis for understanding of the regulation of miRNA on yak CL and also provides a novel genetic resource for future analysis of the gene network during the estrous cycle in the yak.

Caffeic acid is a phenylpropanoid which is widely used in medical industry. Microbial fermentation provides a green strategy for producing caffeic acid. To improve the capacity for caffeic acid production in Escherichia coli, the competing pathways for l-tyrosine synthesis were knocked out. The biosynthesis pathway of the cofactor FAD and the expression of previously reported polyphenol transporters were enhanced to promote the production of caffeic acid. Transcriptomics analysis was conducted to mine potential transporters that could further enhance the titer of caffeic acid in engineered E. coli. Transcriptomics data of E. coli under caffeic acid and ferulic acid stress showed that 19 transporters were upregulated. Among them, overexpression of ycjP, which was previously identified as a sugar ABC transporter permease, improved the caffeic acid titer to 775.7 mg/L. The caffeic acid titer was further improved to 7922.0 mg/L in a 5-L fermenter, the highest titer achieved by microorganisms.

Resveratrol is a polyphenol with numerous applications in food, pharma, and cosmetics. Lack of precursors and low titer are the main problems hindering industrial scale resveratrol production. Based on previous prescreening, expressing the combination of FjTAL, Pc4CL1 and VvSTS achieved the best resveratrol titer. This was further improved to 235.1 mg/L through engineering the shikimic acid pathway, applying a modular enzyme assembly of Pc4CL1 and VvSTS, enhancing p-coumaric acid supply and diverting glycolytic flux toward erythrose-4-phosphate. The titer was increased to 819.1 mg/L following two rounds of multicopy integration of resveratrol biosynthesis and malonyl-CoA supply, respectively. The titer reached 22.5 g/L with a yield on glucose of 65.5 mg/g using an optimum fed-batch strategy in a 5 L bioreactor with morphology control. This research is the highest report on the de novo production of resveratrol in Yarrowia lipolytica and the findings lay a solid foundation for other producing polyphenols.

O-Acetylhomoserine (OAH) is an important platform chemical for the synthesis of L-methamidophos and l-methionine. It has been produced efficiently in Corynebacterium glutamicum. However, a wider range of key factors had not been identified, limiting further increases in OAH production. This study successfully identified some limiting factors and regulated them to improve OAH titer. Firstly, an efficient clustered regularly interspaced short palindromic repeats/dead CRISPR associated protein 9 (CRISPR-dCas9) system was constructed and used to identify the key genes in central metabolism and branch pathways associated with OAH biosynthesis. Then, the gltA gene involved in TCA cycle was identified as the most critical gene. A sequential promoter PNCgl2698, which showed different transcriptional intensity in different strain growth periods, was used to control the expression of gltA gene, resulting in OAH production of 7.0 g/L at 48 h. Finally, the OAH titer of the engineered strain reached 25.9 g/L at 72 h in a 5-L bioreactor. These results show that the identification and regulation of key genes are critical for OAH biosynthesis, which would provide a better research basis for the industrial production of OAH in C. glutamicum.

Polysaccharides are important natural biomacromolecules. In particular, microbial exopolysaccharides have received much attention. They are produced by a variety of microorganisms, and they are widely used in the food, pharmaceutical, and chemical industries. The Candida glabrata mutant 4-C10, which has the capacity to produce exopolysaccharide, was previously obtained by random mutagenesis. In this study we aimed to further enhance exopolysaccharide production by systemic fermentation optimization. By single factor optimization and orthogonal design optimization in shaking flasks, an optimal fermentation medium composition was obtained. By optimizing agitation speed, aeration rate, and fed-batch fermentation mode, 118.6 g L−1 of exopolysaccharide was obtained by a constant rate feeding fermentation mode, with a glucose yield of 0.62 g g−1 and a productivity of 1.24 g L−1 h−1. Scaling up the established fermentation mode to a 15-L fermenter led to an exopolysaccharide yield of 113.8 g L−1, with a glucose yield of 0.60 g g−1 and a productivity of 1.29 g L−1 h−1.

Anthocyanins are natural pigments found in various plants. As multifunctional natural compounds, anthocyanins are widely used in food, pharmaceuticals, health products, and cosmetics. At present, the anthocyanins are heterologously biosynthesized in prokaryotes from flavan-3-ols, which is rather expensive. This study aimed to metabolically engineer Saccharomyces cerevisiae for anthocyanin production. Anthocyanin production has been extensively studied to understand the metabolic pathway enzymes in their natural hosts, including CHS (chalcone synthase); FLS (flavonol synthase); CHI (chalcone isomerase); F3H (flavanone 3-hydroxylase); F3′H (flavonoid 3′-hydroxylase); F3′5′H (flavonoid 3′,5′-hydroxylase); DFR (dihydroflavonol 4-reductase); ANS (anthocyanidin synthase); LAR (leucoanthocyanidin reductase); and UFGT (flavonoid 3-O-glucosyltransferase). The anthocyanin transporter MdGSTF6 was first introduced and proven to be indispensable for the biosynthesis of anthocyanins. By expressing MdGSTF6, FaDFR, PhANS0, and Dc3GT and disrupting EXG1 (the main anthocyanin-degrading enzyme), the BA-22 strain produced 261.6 mg/L (254.5 mg/L cyanidin-3-O-glucoside and 7.1 mg/L delphinidin-3-O-glucoside) anthocyanins from 2.0 g/L dihydroflavonols, which was known to be the highest titer in eukaryotes. Finally, 15.1 mg/L anthocyanins was obtained from glucose by expressing the de novo biosynthesis pathway in S. cerevisiae, which is known to be the highest de novo production. It is the first study to show that through the introduction of a plant anthocyanin transporter and knockout of a yeast endogenous anthocyanin degrading enzyme, the anthocyanin titer has been increased by more than 100 times.

Background Dihydroquercetin (DHQ), a powerful bioflavonoid, has a number of health-promoting qualities and shows potential as a treatment for a number of disorders. Dihydroquercetin biosynthesis is a promising solution to meet the rising demand for dihydroquercetin. However, due to the significant accumulation of eriodietyol (ERI), naringenin (NAR), dihydrokaempferol (DHK), and other metabolites, the yield of DHQ biosynthesis is low. As a result, this is the hindrance to the biosynthesis of DHQ. Results In this study, we proposed several strategies to enhance the product formation and reduce the metabolites in accumulation. The flavonoid 3′-hydroxylase (F3′H) and cytochrome P450 reductase from different species were co-expressed in S. cerevisiae, and the best strain expressing the P450-reductase enzyme complex (SmF3′H/ScCPR) yielded 435.7 ± 7.6 mg/L of ERI from NAR in the deepwell microplate. The product conversion rate was improved further by mutating the predicted potential ubiquitination sites to improve SmF3′H stability, resulting in a 12.8% increase in titre using the mutant SmF3′H (K290R). Besides, different F3Hs from various sources and promoters were tested for the improved DHQ production, with the best strain producing 381.2 ± 10.7 mg/L of DHQ from 1 g/L of NAR, suggesting the temporal regulation the expression of F3H is important for maximization the function of F3′H and F3H. Conclusion This study offers effective strategies for improving DHQ production from NAR and could be used as a reference for related research.

D-Glucose directly generates 2-keto-L-gulonic acid (2-KLG, precursor of vitamin C) through the 2,5-diketo-D-gluconic acid (2,5-DKG) pathway. 2,5-DKG is the main rate-limiting factor of the reaction, and there are few relevant studies on it. In this study, a more accurate quantitative method of 2,5-DKG was developed and used to screen G. oxydans ATCC9937 as the chassis strain for the production of 2,5-DKG. Combining the metabolite profile analysis and knockout and overexpression of production strain, the non-enzymatic browning of 2,5-DKG was identified as the main factor leading to low yield of the target compound. By optimizing the fermentation process, the fermentation time was reduced to 48 h, and 2,5-DKG production peaked at 50.9 g/L, which was 139.02% higher than in the control group. Effectively eliminating browning and reducing the degradation of 2,5-DKG will help increase the conversion of 2,5-DKG to 2-KLG, and finally, establish a one-step D-glucose to 2-KLG fermentation pathway.

Fusion with self-assembling amphipathic peptides (SAPs) could improve enzyme stability and activity in addition to their effects on protein expression. In this study, we proposed a metabolic regulation strategy based on an iterative SAP fusion with key enzymes in pathway. First, an SAP, S1nv10 (ANANARARANANARAR), was separately fused to the N-terminus of metabolite enzymes. To achieve the diversity in the enzyme activities, a library of the fused SAPs varied in hydrophobicity, length and net charge were generated by a multi-primer PCR procedure and the DATEL-assembly method. After simultaneous modification of three enzymes (CrtE, CrtY, and CrtI) using the SAP library, the Escherichia coli strain with 3.91-fold increase in β-carotene production was isolated. As indicated by western-blot and catalytic property analysis, SAP fusion induced the increases in both enzyme expression (CrtY and CrtE) and enzymatic activities (CrtY). The effectiveness of the strategy was further confirmed by the metabolic regulation of the eriodictyol and (2S)-naringenin, the yields of which were increased by 111.3% and 186.3% in E. coli, respectively. These results indicated that fusion with SAPs could effectively regulate metabolic pathway through optimization of the protein expression and catalytic properties of the key enzymes.

Flavonoids have notable biological activities and have been widely used in the medicinal and chemical industries. However, single-copy integration of heterologous pathway genes limits the production of flavonoids. In this work, we designed and constructed single-step integration of multiple flavonoid (2S)-naringenin biosynthetic pathway genes in S. cerevisiae. The efficiency of the naringenin metabolic pathway gene integration into the rDNA site reached 93.7%. Subsequently, we used a high titer p-coumaric acid strain as a chassis, which eliminated feedback inhibition of tyrosine and downregulated the competitive pathway. The results indicated that increasing the supply of p-coumaric acid was effective for naringenin production. We additionally optimized the amount of donor DNA. The optimum strain produced 149.8 mg/L of (2S)-naringenin. The multi-copy integration of flavonoid pathway genes effectively improved (2S)-naringenin production in S. cerevisiae. We further analyzed the copy numbers and expression levels of essential genes (4CL and CHS) in the (2S)-naringenin metabolic pathway by qPCR. Higher copy numbers of the (2S)-naringenin metabolic pathway genes were associated with greater 4CL and CHS transcription, and the efficiency of naringenin production was higher. Therefore, multi-copy integration of genes in the (2S)-naringenin metabolic pathway was imperative in rewiring p-coumaric acid flux to enhance flavonoid production.

Background Streptomyces mobaraenesis transglutaminase (smTG) is widely used to generate protein crosslinking or attachment of small molecules. However, the low thermostability is a main obstacle for smTG application. In addition, it is still hard to achieve the secretory expression of active smTG in E. coli, which benefits the enzyme evolution. In this study, a combined strategy was conducted to improve the thermostability and secretory expression of active smTG in E. coli. Results First, the thermostable S. mobaraenesis transglutaminase variant S2P-S23V-Y24N-S199A-K294L (TGm1) was intracellularly expressed in pro-enzyme form in E. coli. Fusing the pro-region of Streptomyces hygroscopicus transglutaminase (proH) and TrxA achieved a 9.78 U/mL of intracellular smTG activity, 1.37-fold higher than the TGm1 fused with its native pro-region. After in vitro activation by dispase, the TGm1 with proH yielded FRAPD-TGm1, exhibiting 0.95 ℃ and 94.25% increases in melting temperature and half-life at 60 ℃ compared to FRAP-TGm1 derived from the expression using its native pro-region, respectively. Second, the TGm1 with proH was co-expressed with transglutaminase activating protease and chaperones (DnaK, DnaJ, and GrpE) in E. coli, achieving 9.51 U/mL of intracellular FRAPD-TGm1 without in vitro activation. Third, the pelB signal peptide was used to mediate the secretory expression of active TGm in E. coli, yielding 0.54 U/mL of the extracellular FRAPD-TGm1. A script was developed to shuffle the codon of pelB and calculate the corresponding mRNA folding energy. A 1.8-fold increase in the extracellular expression of FRAPD-TGm1 was achieved by the Top-9 pelB sequence derived from the coding sequences with the lowest mRNA folding energy. Last, deleting the gene of Braun’s lipoprotein further increased the extracellular yield of FRAPD-TGm1 by 31.2%, reached 1.99 U/mL. Conclusions The stabilized FRAPD-smTG here could benefit the enzyme application in food and non-food sectors, while the E. coli system that enables secretory expression of active smTG will facilitate the directed evolution for further improved catalytic properties. The combined strategy (N-terminal modification, co-expression with chaperones, mRNA folding energy optimization of signal peptide, and lipoprotein deletion) may also improve the secretory expression of other functional proteins in E. coli.

Streptomyces mobaraensis transglutaminase (TGase) is extracellularly expressed as a zymogen and then activated by TGase-activating protease (TAP). In this study, we reported the strategy for improving TGase production via the regulation of TAP activity in S. mobaraensis. First, we analyzed the effects of three inorganic nitrogen sources on TGase production. With 30 mM nitrogen content, the time to the peak of TGase activity induced by (NH4)2SO4 or NH4Cl was 72 h, 12 h earlier than that of the fermentation without adding NH4+. SDS-PAGE analysis indicated that NH4+ accelerated the TGase activation in S. mobaraensis. Then, we examined the effect of NH4+ on TAP biosynthesis using a TGase-deficient S. mobaraensis strain. It showed that NH4+ enhanced the TAP activity at the early stage of the fermentation, which was dependent on the concentration and time of NH4+ addition. Last, the yield and productivity of S. mobaraensis TGase were increased by 1.18-fold and 2.1-fold, respectively, when optimal NH4+ addition (60 mM and 12 h) was used. The fermentation period was shortened from 84 to 48 h. The NH4+ addition also increased the storage stability of crude enzyme at room temperature. These findings will benefit the TGase production and its activation mechanism in S. mobaraensis.

Pyridoxal 5′-phosphate (PLP) is an essential cofactor that participates in ∼4% enzymatic activities cataloged by the Enzyme Commission. The intracellular level of PLP is usually lower than that demanded in industrial catalysis. To realize the self-supply of PLP cofactor in whole-cell biotransformation, the de novo ribose 5-phosphate (R5P)-dependent PLP synthesis pathway was constructed. The pdxST genes from Bacillus subtilis 168 were introduced into the tyrosine phenol-lyase (TPL)-overexpressing Escherichia coli BL21(DE3) strain. TPL and PdxST were co-expressed with a double-promoter or a compatible double-plasmid system. The 3,4-dihydroxyphenylacetate-L-alanine (L-DOPA) titer did not increase with the increase in the intracellular PLP concentration in these strains with TPL and PdxST co-expression. Therefore, it is necessary to optimize the intracellular PLP metabolism level so as to achieve a higher L-DOPA titer and avoid the formation of L-DOPA–PLP cyclic adducts. The thi riboswitch binds to PLP and forms a complex such that the ribosome cannot have access to the Shine-Dalgarno (SD) sequence. Therefore, this metabolite-sensing regulation system was applied to regulate the translation of pdxST mRNA. Riboswitch was introduced into pET–TPL–pdxST-2 to downregulate the expression of PdxST and biosynthesis of PLP at the translation level by sequestering the ribosome-binding site. As a result, the titer and productivity of L-DOPA using the strain BL21–TPLST–Ribo1 improved to 69.8 g/L and 13.96 g/L/h, respectively, with a catechol conversion of 95.9% and intracellular PLP accumulation of 24.8 μM.

Metabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering. Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor malonyl-CoA with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitions malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing transcriptional activator FdeR and yeast hybrid promoters to control leucine synthesis and cell grwoth fitness, this amino acid feedforward metabolic circuit confers a flavonoid addiction phenotype that selectively enrich the naringenin-producing pupulation in the leucine auxotrophic yeast. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations. Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield in long-term and large-scale bioproduction.

The abundant supply of biosynthetic precursors and product compatibility with the intracellular environment play important roles for microbial isoprenoid production. In this study, we tailor to both of these requirements by introducing the two-step isopentenol utilization pathway (IUP) to augment the native pathway in the oleaginous yeast Yarrowia lipolytica. With shortcut access to the common isoprenoid precursor, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), IUP is capable of elevating IPP + DMAPP levels by 15.7-fold compared to the mevalonate pathway alone. The increase in IPP + DMAPP levels can directly lead to better isoprenoid synthesis, which is illustrated using lycopene as a model compound. Moreover, we also demonstrate that higher lipid contents in the cells correlate with improved intracellular lycopene production, suggesting the importance of having a substantial hydrophobic environment to sequester isoprenoids. Combining these strategies with further genetic and fermentation optimizations, we achieved a final lycopene titer of 4.2 g/L. Overall, these strategies hold great potential for strengthening the synthesis of long-chain isoprenoids and fat-soluble natural products in microbes.

Industrial production of 2-keto-L-gulonic acid (2-KLG), the precursor of vitamin C, is mainly achieved by a two-step fermentation process carried out by Gluconobacter oxydans, Bacillus, and Ketogulonicigenium. One of the most promising innovations that could replace this complicated two-step fermentation process is the integration of the essential genes for synthesis of 2-KLG into G. oxydans and use of it as the producer. Therefore, determining the tolerance and response of G. oxydans to 2-KLG is a priority for improving the direct production of 2-KLG in this bacterium. In this study, a global view of the gene expression of G. oxydans WSH-003 in response to 2-KLG challenge was investigated by RNA sequencing. A total of 363 genes of G. oxydans that were differentially expressed in response to 2-KLG were uncovered. The results showed that 2-KLG could lead to oxidative stress, osmotic stress, and DNA damage in G. oxydans.

The formation of inclusion bodies (IBs) without enzyme activity in bacterial research is generally undesirable. Researchers have attempted to recovery the enzyme activities of IBs, which are commonly known as active IBs. Tyrosine phenol-lyase (TPL) is an important enzyme that can convert pyruvate and phenol into 3,4-dihydroxyphenyl-l-alanine (L-DOPA) and IBs of TPL can commonly occur. To induce the correct folding and recover the enzyme activity of the IBs, peptides, such as ELK16, DKL6, L6KD, ELP10, ELP20, L6K2, EAK16, 18A, and GFIL16, were fused to the carboxyl terminus of TPL. The results showed that aggregate particles of TPL-DKL6, TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16 improved the enzyme activity by 40.9%, 50.7%, 48.9%, 86.6%, and 97.9%, respectively. The peptides TPL-DKL6, TPL-EAK16, TPL-18A, and TPL-GFIL16 displayed significantly improved thermostability compared with TPL. L-DOPA titer of TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16, with cells reaching 37.8 g/L, 53.8 g/L, 37.5 g/L, and 29.1 g/L, had an improvement of 111%, 201%, 109%, and 63%, respectively. A higher activity and L-DOPA titer of the TPL-EAK16 could be valuable for its industrial application to biosynthesize L-DOPA. Electronic supplementary material The online version of this article (10.1007/s10295-020-02294-4) contains supplementary material, which is available to authorized users.

l -Tyrosine is an important amino acid widely used in food, agriculture, and pharmaceutical industries. However, the industrial application was severely constrained due to low production. To obtain the Escherichia coli mutant producing l -tyrosine in abundance, the heat-inducible expression vector carrying the two feedback resistance enzymes (3-deoxy-7-phosphoheptulonate synthase encoded by aroGfbr and chorismate mutase/prephenate dehydrogenase encoded by tyrAfbr) were introduced into the phenylalanine-producing E. coli strain to enable it to synthesize l -tyrosine directly from glucose. Furthermore, the CRISPR-Cas9 technology was applied to eliminate l -phenylalanine and l -tryptophan pathways for their competition for the carbon flux. The global regulatory protein TyrR, which mediates the biosynthesis and transportation of aromatic amino acids, was also deleted to increase l -tyrosine production. Among the recombinant strains, the pheA/tyrR double-gene deletion strain had the highest yield of 5.84 g/L on shake flasks. The feeding strategies were then optimized in a 3-L fermentor. The pheA/tyrR double-gene deletion strain with the heat-inducible expression plasmid pAP-aroGfbr-tyrAfbr was able to produce 55.54 g/L l -tyrosine by fed-batch fermentation; the substrate conversion rate was 0.25 g/g. The recombinant strains constructed in this study could be an industrial platform for the microbial production of l -tyrosine directly from glucose.

3,4-Dihydroxyphenyl-L-alanine (L-DOPA) is the most important antiparkinsonian drug, and tyrosine phenol-lyase (TPL)-based enzyme catalysis process is one of the most adopted methods on industrial scale production. TPL activity and stability represent the rate-limiting step in L-DOPA synthesis. Here, 25 TPL mutants were predicted, and two were confirmed as exhibiting the highest L-DOPA production and named E313W and E313M. The L-DOPA production from E313W and E313M was 47.5 g/L and 62.1 g/L, which was 110.2 % and 174.8 % higher, respectively, than that observed from wild-type (WT) TPL. The Km of E313W and E313M showed no apparent decrease, whereas the kcat of E313W and E313M improved by 45.5 % and 36.4 %, respectively, relative to WT TPL. Additionally, E313W and E313M displayed improved thermostability, a higher melting temperature, and enhanced affinity between for pyridoxal-5′-phosphate. Structural analysis of the mutants suggested increased stability of the N-terminal region via enhanced interactions between the mutated residues and H317. Application of these mutants in a substrate fed-batch strategy as whole-cell biocatalysts allows realization of a cost-efficient short fermentation period resulting in high L-DOPA yield.

L-Homoserine is an important platform compound that is widely used to produce many valuable bio-based products, but production of L-homoserine in Corynebacterium glutamicum remains low. In this study, an efficient L-homoserine-producing strain was constructed. Native pentose phosphate pathway (PPP) was enhanced and heterologous Entner-Doudoroff (ED) pathway was carefully introduced into L-homoserine-producing strain, which increased the L-homoserine titer. Coexpression of NADH-dependent aspartate-4-semialdehyde dehydrogenase and aspartate dehydrogenase could increase the titer from 11.3 to 13.3 g/L. Next, NADP

Lipoxygenase (LOX) catalyzes the peroxidation of unsaturated fatty acids to produce hydroperoxides, which had been widely used in food, medicine and chemical industries due to its decoloration of food and conversion of renewable oils. Thus, higher catalytic activity and stability is desired for low-cost and expanded industrial applications of LOX. To improve the catalytic activity of LOX, a mutant library of Pseudomonas aeruginosa lipoxygenase (PaLOX) was firstly built via semi-rational design. The k

Background Lipoxygenase (LOX) is a non-heme iron containing dioxygenase that is widely used to improve food quality and produce active drug intermediates and biodiesel. Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression; however, its weak extracellular secretion ability precludes its effective production of recombinant proteins into the extracellular environment. To facilitate subsequent characterization and application of LOX, improving its secretion efficiency from E. coli is a major challenge that needs to be solved. Results Several strategies were adopted to improve the extracellular secretion of LOX based on the signal peptides and cell wall permeability of E. coli. Here, we studied the effect of signal peptides on LOX secretion, which increased the secretory capacity for LOX marginally. Although surfactants could increase the permeability of the cell membrane to promote LOX secretion, the extracellular LOX yield could not meet the requirements of industrialization production. Subsequently, an autolysis system was constructed in E. coli based on the bacteriophage lysis gene ΦX174-E to enhance the production of extracellular proteins. Thus, the extracellular production of LOX was achieved and the content of inclusion bodies in the cell was reduced by optimizing cell lysis conditions. The extracellular LOX yield reached 368 ± 1.4 U mL−1 in a 5-L bioreactor under optimized lysis conditions that is, an induction time and temperature, and arabinose concentration of 5 h, 25 °C, and 0.6 mM, respectively. Conclusions In this study, the different signal peptides and cell autolysis system were developed and characterized for extracellular LOX production in E. coli. Finally, the cell autolysis system presented a slight advantage on extracellular LOX yield, which also provides reference for other protein extracellular production.

Scleroglucan is a non-ionic water-soluble polysaccharide, and has been widely used in the petroleum, food, medicine and cosmetics industries. Currently, scleroglucan is mainly produced by Sclerotium rolfsii. A higher level of scleroglucan (42.0 g/L) was previously obtained with S. rolfsii WSH-G01. However, the production of scleroglucan was reduced despite a higher glucose concentration remaining. Additionally, the molecular weight of scleroglucan was large, thus restricted its application. In this study, by adjusting the state of seeds inoculated, the degradation issue of scleroglucan during the fermentation process was solved. By comparing different fed-batch strategies, 66.6 g/L of scleroglucan was harvested by a two-dose fed-batch mode, with 53.3% glucose conversion ratio. To modify the molecular weight of scleroglucan, a combination method with HCl and high-pressure homogenization treatment was established. Finally, scleroglucan with molecular weight of 4.61 × 105 Da was obtained. The developed approaches provide references for the biosynthesis and molecular weight modification of polysaccharides.

Gluconobacter oxydans is important in the conversion of D-sorbitol into l-sorbose, which is an essential intermediate for industrial-scale production of vitamin C. In a previous study, the strain G. oxydans WSH-004 could directly produce 2-keto-l-gulonic acid (2-KLG). However, its D-sorbitol tolerance was poor compared with that of other common industrial G. oxydans strains, which grew well in the presence of more than 200 g/L of D-sorbitol. This study aimed to use the microbial microdroplet culture (MMC) system for the adaptive evolution of G. oxydans WSH-004 so as to improve its tolerance to high substrate concentration and high temperature. A series of adaptively evolved strains, G. oxydans MMC1-MMC10, were obtained within 90 days. The results showed that the best strain MMC10 grew in a 300 g/L of D-sorbitol medium at 40°C. The comparative genomic analysis revealed that genetic changes related to increased tolerance were mainly in protein translation genes. Compared with the traditional adaptive evolution method, the application of microdroplet-aided adaptive evolution could improve the efficiency in terms of reducing time and simplifying the procedure for strain evolution. This research indicated that the microdroplet-aided adaptive evolution was an effective tool for improving the phenotypes with undemonstrated genotypes in a short time.

Flavonoids are a group of valuable compounds with a variety of health benefits. (2 S)-Naringenin is an important flavonoid skeleton, which can be tailored into almost all flavonoids. In this study, the Saccharomyces cerevisiae native precursor pathways were explored and higher-active CHSs from plants rich in flavonoids were screened. The results indicated that overexpressing the native precursor pathways is not an efficient approach to improving (2 S)-naringenin production in our chassis strain. On the other hand, by screening from plants rich in flavonoids, we obtained four CHSs with higher activities than the commonly used PhCHS. Among these CHSs, SjCHS1 increased the (2 S)-naringenin titer by 48.38% in shaking flasks. Finally, we combined the native precursor pathways optimization with the higher-active CHS that screened, and further increased the (2 S)-naringenin titer to 203.49 mg/L from glucose in shaking flasks. The results achieved in this study indicated that plants rich in flavonoids are good sources for higher-active CHS screening, and that the heterologous pathway and chassis precursor flux should be synergistically engineered to achieve higher production.

l-DOPA is a key pharmaceutical agent for treating Parkinson’s, and market demand has exploded due to the aging population. There are several challenges associated with the chemical synthesis of l-DOPA, including complicated operation, harsh conditions, and serious pollution. A biocatalysis route for l-DOPA production is promising, especially via a route catalyzed by tyrosine phenol lyase (TPL). In this study, using TPL derived from Erwinia herbicola (Eh-TPL), a mutant Eh-TPL was obtained by integrating enzyme evolution and high-throughput screening methods. l-DOPA production using recombinant Escherichia coli BL21 (DE3) cells harbouring mutant Eh-TPL was enhanced by 36.5% in shake flasks, and the temperature range and alkali resistance of the Eh-TPL mutant were promoted. Sequence analysis revealed two mutated amino acids in the mutant (S20C and N161S), which reduced the length of a hydrogen bond and generated new hydrogen bonds. Using a fed-batch mode for whole-cell catalysis in a 5 L bioreactor, the titre of l-DOPA reached 69.1 g L−1 with high productivity of 11.52 g L−1 h−1, demonstrating the great potential of Eh-TPL variants for industrial production of l-DOPA.

2-Keto-L-gulonic acid (2-KLG) is the direct precursor of vitamin C in industrial synthesis. 2-KLG is mainly produced via the classical two-step fermentation route. In the two-step fermentation process, 2-KLG can be synthesized from L-sorbose by Ketogulonicigenium vulgare aided by Bacillus megaterium. There are five sorbose/sorbosone dehydrogenases (SSDHs), SSDA1, SSDA1-P, SSDA2, SSDA3 and SSDB, and two sorbosone dehydrogenases (SNDHs), glucose/sorbosone dehydrogenase (GSNDH) and sorbosone dehydrogenase (SNDH), in K. vulgare, which could play crucial roles in transforming L-sorbose or L-sorbosone to 2-KLG. However, confusion about the catalytic characteristics of the individual SSDHs and SNDHs makes construction of a recombinational strain for the purpose of enhancing 2-KLG production difficult. In this study, the five SSDHs and two SNDHs from K. vulgare WSH-001 were purified, and their optimal pH values and reaction temperatures, kinetic properties, thermostabilities, substrate spectra and effects of electron acceptors on their performances were systematically determined. Among these dehydrogenases, only SSDA1 and SSDA3 have high activity for catalyzing L-sorbose to 2-KLG directly. These data provide more clues for ways to achieve enhanced conversion of L-sorbose in K. vulgare, which could facilitate both the construction of a more efficient one-step fermentation 2-KLG producer and the reconstruction of a one-step fermentation process.

Acetic acid bacteria (AAB) are a group of gram-negative, obligate aerobic bacteria within the Acetobacteraceae family of the alphaproteobacteria class, which are distributed in a wide variety of different natural sources that are rich in sugar and alcohols, as well as in several traditionally fermented foods. Their versatile capabilities are not limited to producing acetic acid and brewing vinegar, as their names suggest. They can also be used for fixing nitrogen, yielding pigments and exopolysaccharides (EPS), and most typically, producing a variety of aldehydes, ketones and other organic acids from the incomplete oxidation of the corresponding alcohols and/or sugars (also referred to as oxidative fermentation). In order to gain more insight into these organisms, molecular biology techniques have been extensively applied in almost all aspects of AAB research, including their identification and classification, acid resistance mechanisms, oxidative fermentation, EPS production, thermotolerance and so on. In this review, we mainly focus on the application of molecular biological technologies in the advancement of research into AAB while presenting the progress of the latest studies using these techniques.

Acetic acid bacteria (AAB) are a group of bacteria that can oxidize many substrates such as alcohols and sugar alcohols and play important roles in industrial biotechnology. A majority of industrial processes that involve AAB are related to their dehydrogenases, including PQQ/FAD-dependent membrane-bound dehydrogenases and NAD(P)+-dependent cytoplasmic dehydrogenases. These cofactor-dependent dehydrogenases must effectively regenerate their cofactors in order to function continuously. For PQQ, FAD and NAD(P)+ alike, regeneration is directly or indirectly related to the electron transport chain (ETC) of AAB, which plays an important role in energy generation for aerobic cell growth. Furthermore, in changeable natural habitats, ETC components of AAB can be regulated so that the bacteria survive in different environments. Herein, the progressive cascade in an application of AAB, including key dehydrogenases involved in the application, regeneration of dehydrogenase cofactors, ETC coupling with cofactor regeneration and ETC regulation, is systematically reviewed and discussed. As they have great application value, a deep understanding of the mechanisms through which AAB function will not only promote their utilization and development but also provide a reference for engineering of other industrial strains.

Malonyl-CoA is an important building block for microbial synthesis of numerous pharmaceutically interesting or fatty acid-derived compounds including polyketides, flavonoids, phenylpropanoids and fatty acids. However, the tightly regulated intracellular malonyl-CoA availability often impedes overall product formation. Here, in order to unleash this tightly cellular behavior, we present evolution: dual dynamic regulations-based approaches to write artificial robust and dynamic function into intricate cellular background. Firstly, a conserved core domain based evolutionary principles were incorporated into genome mining to explore the biosynthetic diversities of discrete acetyl-CoA carboxylase (ACC) families, as malonyl-CoA is solely derived from carboxylation of acetyl-CoA by ACC in most organisms. A comprehensive phylogenomic and further experimental analysis, which included genomes of 50 strains throughout representative species, was performed to recapitulate the evolutionary history and reveal that previously unnoticed ACC families from Salmonella enterica exhibited the highest activities among all the candidates. A set of orthogonal and bi-functional quorum-sensing (QS)-based regulation tools were further designed and connected with T7 RNA polymerase as genetic amplifier to achieve dual dynamic control in a high dynamic range, which allowed us to efficiently activate and repress different sets of genes dynamically and independently. These genetic circuits were then combined with ACC of S. enterica and CRISPRi system to reprogram central metabolism that rewired the tightly regulated malonyl-CoA pathway to a robust and autonomous behavior, leading to a 29-fold increase of malony-CoA availability. We applied this dual regulation tool to successfully synthesizing malonyl-CoA-derived compound (2S)-naringenin, and achieved the highest production (1073.8 mg/L) reported to date associate with dramatic decreases of by-product formation. Notably, the whole fermentation presents as an autonomous behavior, totally eliminating human supervision and inducer supplementation. Hence, the constructed evolution: dual dynamic regulations-based approaches pave the way to develop an economically viable and scalable procedure for microbial production of malonyl-CoA derived compounds.

Bacillus licheniformis α-amylase is a thermostable enzyme used in industrial starch hydrolysis. However, difficulties in the genetic manipulation of B. licheniformis hamper further enhancement of α-amylase production. In this regard, adaptive evolution is a useful strategy for promoting the productivity of microbial hosts, although the success of this approach requires the application of suitable evolutionary stress. In this study, we designed a growth-coupled adaptive evolution model to enrich B. licheniformis strains with enhanced amylase productivity and utilization capacity of starch substrates. Single cells of high α-amylase-producing B. licheniformis were isolated using a droplet-based microfluidic platform. Clones with 67% higher α-amylase yield were obtained and analyzed by genome resequencing. Our findings confirmed that growth-coupled evolution combined with high-throughput screening is an efficient strategy for enhanced α-amylase production. In addition, we identified several potential target genes to guide further modification of the B. licheniformis host for efficient protein expression.

The acetic acid bacterium Gluconobacter oxydans is known for its unique incomplete oxidation and therefore widely applied in the industrial production of many compounds, e.g., 2-keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C. However, few molecular tools are available for metabolically engineering G. oxydans, which greatly limit the strain development. Promoters are one of vital components to control and regulate gene expression at the transcriptional level for boosting production. In this study, the low activity of SDH was found to hamper the high yield of 2-KLG, and enhancing the expression of SDH was achieved by screening the suitable promoters based on RNA sequencing data. We obtained 97 promoters from G. oxydans’s genome, including two strong shuttle promoters and six strongest promoters. Among these promoters, P3022 and P0943 revealed strong activities in both Escherichia coli and G. oxydans, and the activity of the strongest promoter (P2703) was about threefold that of the other reported strong promoters of G. oxydans. These promoters were used to overexpress SDH in G. oxydans WSH-003. The titer of 2-KLG reached 3.7 g/L when SDH was under the control of strong promoters P2057 and P2703. This study obtained a series of gradient promoters, including two strong shuttle promoters, and expanded the toolbox of available promoters for the application in metabolic engineering of G. oxydans for high-value products.

Bioproduction of 5-aminovalerate (5AVA) from renewable feedstock can support a sustainable biorefinery process to produce bioplastics, such as nylon 5 and nylon 56. In order to achieve the biobased production of 5AVA, a 2-keto-6-aminocaproate-mediated synthetic pathway was established. Combination of L-Lysine α-oxidase from Scomber japonicus, α-ketoacid decarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli could achieve the biosynthesis of 5AVA from biobased L-Lysine in E. coli. The H2O2 produced by L-Lysine α-oxidase was decomposed by the expression of catalase KatE. Finally, 52.24 g/L of 5AVA were obtained through fed-batch biotransformation. Moreover, homology modeling, molecular docking and molecular dynamic simulation analyses were used to identify mutation sites and propose a possible trait-improvement strategy: the expanded catalytic channel of mutant and more hydrogen bonds formed might be beneficial for the substrates stretch. In summary, we have developed a promising artificial pathway for efficient 5AVA synthesis.

The booming demand for a sustainable future food supply has fueled the development of plant-based meat alternatives (PBMAs) to address the challenges of natural resource depletion, growing populations, and climate change. Innovations and technologies are essential for promoting research and industry growth on PBMAs. In this review, we examine microfluidic technology as a powerful tool that offers solutions for removing manufacturers' hurdles by providing meat replacement products that match customers' expectations and discuss its potential to bring PBMAs to the mainstream through high-throughput screening applications, food structure design, and food safety detection and diagnosis. Finally, prospects and challenges for using microfluidics in the research and industrial production of PBMAs are presented.

The direct fermentation of the precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG), has been a long-pursued goal. Previously, a strain of Gluconobacter oxydans WSH-004 was isolated that produced 2.5 g/L 2-KLG, and through adaptive evolution engineering, the strain G. oxydans MMC3 could tolerate 300 g/L D-sorbitol. This study verified that the sndh-sdh gene cluster encoded two key dehydrogenases for the 2-KLG biosynthesis pathway in this strain. Then G. oxydans MMC3 further evolved through adaptive evolution to G. oxydans 2-KLG5, which can tolerate high concentrations of D-sorbitol and 2-KLG. Finally, by increasing the gene expression levels of the sndh-sdh and terminal oxidase cyoBACD in G. oxydans 2-KLG5, the 2-KLG accumulation in the 5-L fermenter increased to 45.14 g/L by batch fermentation. The results showed that combined evolutionary and metabolic engineering efficiently improved the direct production of 2-KLG from D-sorbitol in G. oxydans.

The 2-keto-L-gulonic acid (2-KLG) is the direct precursor for industrial vitamin C production. The main biosynthetic method for 2-KLG production is the classical two-step fermentation route. However, disadvantages of this method are emerging, including high consumption of energy, difficulties in strain screening, complex operation, and poor stability. In this study, five recombinant Escherichia coli strains overexpressing different sorbose/sorbosone dehydrogenases were constructed and used for 2-KLG production. By optimizing catalytic conditions and further expressing pyrroloquinoline quinone in the recombinant strain, the titer of 2-KLG reached 72.4 g/L, with a conversion ratio from L-sorbose of 71.2% in a 5-L bioreactor. To achieve direct biosynthesis of 2-KLG from D-sorbitol, a co-culture system consisting of Gluconobacter oxydans and recombinant E. coli was designed. With this co-culture system, 16.8 g/L of 2-KLG was harvested, with a conversion ratio from D-sorbitol of 33.6%. The approaches developed here provide alternative routes for the efficient biosynthesis of 2-KLG.